Appl Microbiol Biotechnol
supplemented with 50 μM of each compound to be tested
0.5% v/v DMSO was used as control) and 0.5 or 1 mM hy-
drogen peroxide (H O ). To examine alleviation of oxidative
10 min, the samples were centrifuged for 15 min at 13,000×g at
4 °C and the pellet was washed with cold acetone; this washing
step was repeated for three times. Pellets were dried in under a
vacuum pump, then resuspended in rehydration buffer [8 M
urea, 2% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate hydrate (CHAPS)] and stored at −80 °C until
further use. Total proteins content was determined according to
the Bradford protein assay (Bradford 1976), using bovine se-
rum albumin as standard.
(
2
2
stress of vanillic, gallic, and dihydrolipoic acids; glutathione
GSH); and thiosemicarbazones (all supplemented at the con-
centration of 50 μM) on cells, 0.5 or 1 mM hydrogen peroxide
H O ) was incorporated into the medium. Cell growth was
(
(
2
2
monitored at 28 °C for 7 days. Each assay was performed in
triplicate. Compounds were considered to have anti-oxidant
activity if cell growth improved compared to cohorts exposed
to H O without the test compound.
Proteomic analysis
2
2
Petite frequency assay
Analysis of differentially expressed proteins in A. flavus
mHtcum-treated cultures was conducted by two-
dimensional electrophoresis (2D PAGE), as described in detail
in the online supplementary material. Briefly, 125 μg of total
proteins was loaded on 7-cm-long, pH interval 3–10, strips,
isoelectrofocused and separated in a 12% polyacrylamide gels
(second-dimension run). Gels were stained with SYPRO®
Ruby Protein Stain (BioRad, Hercules, CA, USA); 2-D gel
image elaboration and analysis was carried out with the
PDQuest software version 8.0.1 (BioRad, Hercules, CA,
USA). Three technical replicates for each of the three biolog-
ical replicates were performed, for a total of nine gels for each
class (control and mHtcum-treated samples). Spots differen-
tially expressed were manually removed from the gels, sub-
jected to an in-gel digestion, and addressed to a liquid chro-
matography–electrospray ionization–mass spectrometry (LC-
ESI-MS/MS) and LTQ-Orbitrap analyses. Mass data were
submitted to database searching using SEQUEST search en-
gine Proteom Discoverer interface (Thermo Scientific, version
1.4; Thermo Fisher Scientific, Waltham, MA, USA).
To determine the effect of mHtcum on the yeast mitochondrial
DNA (mtDNA) mutability, W303-1B and DWM-5A strains har-
Y757
G807R
boring mip1
and mip1
alleles, respectively, were pre-
grown on solid synthetic complete medium [SC; 6.9% (w/v)
™
YNB (Formedium , Swaffham, King’s Lynn, Norfolk, UK),
0.1% (w/v) dropout mix] according to Baruffini et al. (2010)
supplemented with 2% (v/v) ethanol at 28 °C to counterselect
the petite cells that could be present in the population. After
approximately 60 h, strains were grown on liquid SC medium
supplemented with 2% (w/v) glucose and mHtcum (50 μM) or
dihydrolipoic acid (50 μM) for two 24-h growth cycles. Control
experiment in which equal amounts of DMSO (0.5% v/v) or
ethanol (0.1% v/v)—the solvents used to solubilize mHtcum
and dihydrolipoic acid, respectively—were added to untreated
cells was done in parallel. Cells were then counted, diluted, and
plated (200–250 cells/plate) on SC medium supplemented with
0.3% (w/v) glucose and 2% (v/v) ethanol. Petite frequency was
defined as the percentage of colonies showing the petite pheno-
type after a 5-day incubation at 28 °C. Each experiment was
repeated at least three times on two independent clones for each
strain. Statistical analysis of petite frequency was performed by a
two-tailed Z test.
RNA extraction and gene expression analysis (quantitative
real-time PCR)
Total RNA was extracted from 96-h-old microplate CCM cul-
®
Preparation of A. flavus total protein extracts
tures using TRIzol Kit (Sigma-Aldrich, Saint Louis, MO,
USA), according to the manufacturer’s instructions. Two-
hundred micrograms of mycelium was flash frozen in liquid
nitrogen, ground to a powder with an amalgamator (TAC 200/
S, Linea TAC s.r.l., Asti, Italy; oscillation frequency 4.200 p/m),
Mycelium (conidia were incubated for 4 days in the same cul-
ture conditions used for aflatoxin determination) was manually
detached from wells and rinsed in ultrapure distilled water.
Samples (200 mg each) were weighed into a prepared tube with
®
and overlaid with 300 μl of TRIzol reagent. The RNA quality
2
00 μl of glass microbeads, frozen in liquid nitrogen, ground
into a powder with the action of a dental amalgamator (TAC
00/S, Linea TAC s.r.l., Montegrosso d’Asti, Italy), and added
was confirmed by gel electrophoresis (1.5% agarose) and con-
centrations were measured using a BioPhotometer (Eppendorf,
Hamburg, Germany). The A260/A280 ratio was measured. Two
micrograms of total RNA sample was reverse-transcribed using
the Maxima First-Strand cDNA Synthesis Kit for qRT-PCR with
dsDNase (Thermo Fisher Scientific, Waltham, MA, USA), fol-
lowing the manufacturer instructions. The complementary DNA
(cDNA) samples were used as templates of qPCR reactions con-
ducted with ABI 7300 instrumentation ((Thermo Fisher
Scientific, Waltham, MA, USA) and SYBR® Green PCR
2
with 200 μl of lysis buffer [50 mM Tris-HCl pH 7.5, 2 M
thiourea, 7 M urea, 2% (v/v) Triton X-100, 1% dithiothreitol
(
1
DTT), 2% (w/v) soluble polyvinylpolypyrrolidone (PVPP),
mM phenylmethylsulphonylfluoride (PMSF), and 0.2% (v/
v) β-mercaptoethanol]. Samples were centrifuged twice for
0 min at 13,000×g, and then, 200 μl 45% (w/v) trichloroacetic
acid was added to the supernatant. After incubation on ice for
2
Degola, Francesca
